Abstract
Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.
Highlights
Streptococcus pneumoniae serogroup 10 includes four crossreactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C)
Linkage analysis identified three times more terminal Galf from intact CPS10B than from the hydrofluoric acid (HF)-treated polysaccharide sample, which suggested the presence of Galf branches. 3-Linked Galp was detected from intact CPS10B, whereas terminal Galp was detected for the HF-treated sample, which suggested a phosphodiester or furanoside linkage to the 3-OH of Galp. 3,4-Linked GalNAc was detected from the intact polysaccharide, and 4-linked GalNAc was detected from the HF-treated sample, which suggested a phosphodiester or furanoside linkage to the 3-OH of GalNAc
Linkage analysis of CPS10C gave results similar to those described for CPS10B, with the exception that 4,6-substituted GalNAc was detected from intact CPS10C versus 4-linked GalNAc from the HF-treated sample, which suggested a phosphodiester or furanoside linkage to the 6-OH of GalNAc
Summary
Streptococcus pneumoniae serogroup 10 includes four crossreactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). The structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype Both CPS10C and CPS10F had 1– 6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent ␣1–2 or wcrF-dependent ␣1– 4 linkages between Gal and ribitol-5-phosphate. Deletion of this gene eliminated wcrG-dependent 1– 6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes. Strain 10061/38 with ermAM in place of wcjG Strain 10061/38 with ermAM in place of wcrG Strain 10061/38 with ermAM in place of wcrD Strain 423/82 with ermAM in place of wcjG Strain 423/82 with ermAM in place of wcrG Strain 423/82 with ermAM in place of wcrD
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